This invention relates to adenovirus vectors, and to methods for making and using such vectors. More particularly, this invention relates to improved adenovirus shuttle vectors containing mutations in the E1B region which permit the deletion of the entire region, or select genes therein, and, optionally substitution with a desired heterologous DNA sequence.
Adenoviruses are a family of viruses that cause benign respiratory tract infections in humans. Over forty-five serotypes have been identified. Strains 5 and 2 from the C subgroup are two strains that have been used to make vectors because their molecular composition is well characterized, although clinical experience with adenoviral vaccines is with strains 4, 7 and 21 (see, for example, Takafuji, J. Inf. Dis. 140: 48-53 (1979)). Adenoviruses are nonenveloped, icosohedral, double-stranded DNA viruses. The genome is about 36 kb in size and codes for over 40 variously sized polypeptides, including early and late proteins. After binding to a target cell through a capsid fiber protein, the virus is internalized into endosomes, with the capsid eventually escaping relatively intact as it migrates to the nucleus. At the nuclear pore, the capsid dissociates and, the viral DNA moves to the cell nucleus and the early proteins are transcribed, resulting in DNA replication and transcription of the late genes that encode the capsid proteins. Replication occurs in the nucleus of the cell typically, although not exclusively, without integration into the host DNA. New viral particles are assembled in the nucleus and the host cells are lysed, about 36 hours post-infection, releasing the virus. Adenoviruses infect a broad range of cell types and transduction has been observed in replicating and in nonreplicating cells.
In recent years, human adenoviruses have become popular as vehicles for gene transfer into mammalian cells because the virus uses the machinery of the host cell to synthesize viral DNA, RNA and proteins and because gene transcription, genomic organization and the identity of the DNA in adenovirus have been well defined. Adenoviral vectors are known and their use facilitates the study of protein function in mammalian cells and permits production of gene products. In addition, adenoviral vectors may be used as recombinant viral vaccines. The usual method of making an adenoviral vector is to substitute the E1 region of the viral genome with a transgene driven by a selected exogenous promoter, normally containing a strong enhancer. Substitutions and/or deletions have also been made in the E3 and E4 regions. Substitutions in the E1 region are particularly advantageous because the E1 proteins have oncogenic and lytic properties.
Modification of the viral genome can be accomplished by employing recombination or by employing molecular biological techniques. The latter approach typically takes advantage of a single Cla-1 site at the left end of the adenovirus strain 5 (xe2x80x9cAd-5xe2x80x9d) genome that disrupts the E1A region, and the left terminal sequences necessary for encapsidation and containing an origin of replication, about 900 basepairs of the 5xe2x80x2 end of the genome. A plasmid expression vector is also constructed having a single Cla-1 site downstream of the desired transgene and a copy of the adenovirus terminal sequences upstream. This vector is cut and ligated to the E1 negative adenoviral fragment and transfected into human 293 kidney cells immortalized by the constitutive expression of E1A and E1B proteins, to provide E1 functions in trans. Plaques are selected and screened for recombinants, repurified and then used to generate bulk stock of purified recombinant adenoviral vector. Yields can be as high as 1012 to 1013 particles/mL. See Stratford-Perricaudet, J. Clin. Invest. 90: 626-30 (1992). However, this approach poses several problems, which are discussed in PCT Patent Publication No. WO 96/25507, published Aug. 22, 1996 (International Application No. PCT/US96/02336).
In another approach, foreign genes are inserted into the E1 region (see McGrory, Virology 163: 614-17 (1988)), into the E3 region (see Hanke, Virology 177: 437-44 (1990) and Bett, J. Virol. 67: 5911-21 (1993)) or into the E3 region of an E1 deleted vector. Briefly, this approach employs replacement of adenoviral genomic DNA sequences with another DNA fragment to create a plasmid containing a genomic sequence which exceeds the packaging limit of the adenovirus virions. The xe2x80x9cconstructxe2x80x9d can then be propagated as a plasmid (pJM17), which can be rescued as infectious virions when the foreign fragment is replaced by homologous recombination with another DNA fragment small enough to permit packaging. For additional general information on these vectors and their uses see, Hitt, Construction and propagation of human adenovirus vectors. In: Cell Biology: a Laboratory Handbook, J. Celis (ed), Academic Press, N.Y, 1995; see Graham, Adenovirus based expression vectors and recombinant vacines. In: Vaccines: New Approaches to Immunological Problems. R. W. Ellis (ed), Butterworth, pp 363-390, 1992 and see Graham, Manipulation of adenovirus vectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Techniques. E. J. Murry and J. M. Walker (eds) Humana Press Inc., Clifton, N.J., pp 109-128, 1991.
The originally designed and constructed adenovirus vectors reduced virus replication by deleting or mutating portions of the E1A and E1B region. The disadvantage to this approach was that packaging into viral capsids was inefficient, due to the increase in genome size from the addition of the transgene, and the viral stock titer was concomitantly reduced. Deletions in the E3 region increased the amount of heterogeneous DNA which could be inserted into the vector, but it was believed that expression of the E3 gene was needed to assist virus infected cells in avoiding the host immune response.
Thus, it would be advantageous to provide an improved adenovirus vector permitting large insertions of heterogeneous DNA without sacrificing packaging efficiency or protective immunity.
In one aspect, the invention provides methods for constructing replication competent, adenoviral shuttle vectors that can have one or more genes in the E1B region deleted, the vectors so constructed, and optionally heterologous genes of interest substituted for the deleted genes which allows for the temporal expression of the heterologous genes in a manner similar to the endogenous adenoviral gene it replaces.
In a second aspect, the invention provides an E1B shuttle vector wherein the 55K adenoviral gene can be deleted and a heterologous gene of interest substituted therefore.
In another aspect, the invention provides an E1B shuttle vector wherein the E1B region genes, 19K, and 55K can be deleted, and a heterologous gene of interest substituted therefore.
In another aspect, the invention provides an E1B shuttle vector wherein either the 19K, 55K and pIX genes can be deleted, or the pIX gene can be deleted, and a heterologous gene of interest substituted therefore.
In a further aspect of the invention, the E1B shuttle vectors may express a heterologous gene, which expression is preferably driven by the endogenous E1B promoter, wherein the gene encodes a cytokine, including the interleukins, tumor necrosis factor alpha, interferon gamma, or cell cycle regulatory proteins, including p16, or ras, or proteins that induce cellular suicide, or apoptosis, or prodrug activators, including cytosine deaminase or thymidine kinase, or a chemokine including macrophage inhibitory proteins (mip), including mip-3 alpha. The temporal expression pattern of the heterologous gene is similar to the endogenous adenoviral gene it replaces.
In yet another aspect of the invention, E1B shuttle vectors are described wherein such vectors also have mutations elsewhere in the adenoviral genome, preferably in the E1A, E3 and/or E4 regions.
In another aspect of the invention, methods are described wherein the invention E1B shuttle vectors are used for the beneficial treatment or prevention of disease.
In a further aspect of the invention, E1B shuttle vectors may be administered to a patient in a therapeutically effective amount and in a pharmaceutically acceptable carrier. These and other objects of the present invention will become apparent to one of ordinary skill in the art upon reading the description of the various aspects of the invention in the following specification. The foregoing and other aspects of the present invention are explained in greater detail in the drawings, detailed description, and examples set forth below.